A number of mammalian glucose (hexose) transporters (GLUTs have been identified. High affinity GLUTs are found in nearly every tissue. A low affinity GLUT (GLUT-2) is expressed in tissues which are associated with high glucose flux (e.g., intestine, kidney, and liver). It is thought that the level of expression of high affinity GLUTs influences the rate or glucose uptake. It is also thought that the expression of various GLUTs is regulated by glucose and various hormones (Thorens, Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33:G541-G553, 1996). Human GLUT-1 is described by Mucckler et al. (Science 229:941, 1985). Human GLUT-2 is described by Fukumoto et al. (Proc. Nat""l Acad. Sci. USA 264:776, 1989). Human GLUT-3 is described by Keller et al. (J. Biol. Chem. 264:18884, 1989). Human GLUT-4 is described by Fukumoto et al. (J. Biol. Chem. 264:7776, 1989). Human GLUT-5 is described by Kayano et al. (Nature 377:151, 1995).
The invention described herein relates discovery and characterization of a cDNA encoding GLUTX, a human glucose transporter protein. The nucleotide sequence of a cDNA encoding GLUTX is shown in FIGS. 1A-1E. The deduced amino acid sequence of GLUTX is shown in FIGS. 2A-2D. GLUTX is predicted to include 12 transmembrane domains. The first transmembrane domain extends from about amino acid 52 (intracellular end) to about amino acid 71 (extracellular end). The second transmembrane domain extends from about amino acid 108 (extracellular end) to about amino acid 128 (intracellular end). The third transmembrane domain extends from about amino acid 141 (intracellalar end) to about amino acid 159 (extracellular end). The fourth transmembrane domain extends from about amino acid 166 (extracellular end) to about amino acid 189 (intracellular end). The fifth transmembrane domain extends from about amino acid 204 (intracellular end) o about amino acid 221 (extracellular end). The sixth transmembrane domain extends from about amino acid 233 (extracellular end) to about amino acid 252 (intracellular end). The seventh transmembrane domain extends from about amino acid 317 (intracellular end) to about amino acid 338 (extracellular end). The eighth transmembrane domain extends from about amino acid 355 (extracellular end) to about amino acid 375 (intracellular end). The ninth transmembrane domain extends from about amino acid 383 (intracellular end) to about amino acid 404 (extracellular end). The tenth transmembrane domain extends from about amino acid 413 (extracellular end) to about amino acid 437 (intracellular end). The eleventh transmembrane domain extends from about amino acid 449 (intracellular end) to about amino acid 472 (extracellular end). The twelfth transmembrane domain extends from about amino acid 481 (extracellular end) Ho about amino acid 499 intacelllar end). GLUTX nucleic acids and polypeptides, as well as molecules which increase or decrease expression or activity of GLUTX, are useful in the diagnosis and treatment of disorders associated with aberrant hexose transport.
GLUTX protein has some sequence similarity to a number of known glucose transporters FIGS. 3A-3D.
The invention features isolated nucleic acid molecules (i.e., a nucleic acid molecule that is separated from the 5xe2x80x2 and 3xe2x80x2 coding sequences with which to is immediately contiguous in the naturally occurring genome of an organism, also referred to as a recombinant nucleic acid molecule) that encodes a GLUTX Collectide. Within the invention are polypeptides having the sequence of SEQ ID NO:2 or encoded by nucleic acid molecules having the sequence shown in SEQ ID NO:1. However, the invention is not limited to nucleic acid molecules and polypeptides that are identical to those SEQ ID Nos. For example, the invention includes nucleic acid molecules which encode splice variants, allelic variants or mutant forms of GLUTX as well as the proteins encoded by such nucleic acid molecules.
Also within the invention are nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NO:1. Such molecules include, for example, nucleic acid molecules encoding allelic variants of GLUTX or mutant forms of GLUTX. As described further below, molecules that are substantially identical to those of SEQ ID Nos. 1 and 2 are also encompassed by the invention.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given compound (e.g., a GLUTX polypeptide) means that the compound is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the compound may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). When chemically synthesized, a compound of the invention is substantially pure when it is substantially free from the chemical compounds used in the process of its synthesis. Polypeptides or other compounds of interest are substantially free from other compounds when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferable at east 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that s completely identical o a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
For polypeptides, the length of the reference polypeptide sequence will generally be at least it amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleocides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides (e.g., 150, 200, 250, or 300 nucleotides).
In the case of polypeptide sequences that are less than 100% Identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 with the default parameters as specified therein.
The BLAST programs, provided as a service by the National Center for Biotechnology Information, are very useful for making sequence comparisons. The programs are described in detail by Karlin et al., (Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 and 90:5873-7, 1993) and Altschul et al., (Nucl. Acids Res. 25:3389-3402, 1997) and are available on the internet.
The invention also features a host cell that harbors an isolated nucleic acid molecule encoding GLUTX (either alone or in conjunction with a heterologous polyptide, such as a detectable marker) or a nucleic acid vector that contains a sequence encoding GLUTX (again, with or without a heterologous polypeptide). The vector can be an expression vector, and the expression vector can include a regulatory element. An antibody that specifically binds a GLUTX polypeptide is also within the scope of the present invention and is useful, for example, to detect GLUTX in a biological sample or to alter the activity of GLUTX. For example, GLUTX can be detected in a biological sample by contacting the sample with an antibody that specifically binds GLUTX under conditions that allow the formation of a GLUTX-antibody complex and detecting the complex, if present, as an indication of the presence of GLUTX in the sample. The use of an antibody in a treatment regime, where it can alter the activity of GLUTX, is discussed further below.
An antibody of the invention can be a monoclonal, polyclonal, or engineered antibody that specifically binds GLUTX as described more fully below) An antibody that xe2x80x9cspecifically bindsxe2x80x9d to a particular antigen, for example, a GLUTX polypeptide of the invention, will not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, that includes GLUTX.
Given that an object of the present invention is to alter the expression or activity of GLUTX in vivo, a pharmaceutical composition containing, for example, an isolated nucleic acid molecule encoding GLUTX or a fragment thereof), a nucleic acid molecule that is antisense to GLUTX (i.e., that has a sequence that is the reverse and complement of a portion of the coding strand of a GLUTX gene), a GLUTX polypeptide, or an antibody, small molecule, or other compound that specifically binds a GLUTX polypeptide is also a feature of the invention.
The discovery and characterization of GLUTX and the polypeptide it encodes makes it possible to determine whether a given disorder is associated with aberrant expression of GLUTX either at he transcriptional or translational level) or activity of GLUTX. For example, one can diagnose a patient as having a disorder associated with aberrant expression of GLUTX by measuring GLUTX expression in a biological sample obtained from the patient. An increase or decrease in GLUTX expression in the biological sample, compared with GLUTX expression in a control sample (e.g., a sample of the same tissue collected from one or more healthy individuals) indicates that the patient has a disorder associated with aberrant expression of GLUTX. Similarly, one can diagnose a patient as having a disorder associated with aberrant activity of GLUTX by measuring GLUTX activity n a biological sample obtained from the patient. An increase or decrease in GLUEX activity in the biological sample, compared with GLUTX activity in a control sample, indicates that the patient has a disorder associated with aberrant activity of GLUX. The techniques required to measure gene expression or polypeptide activity are well known to those of ordinary skill in the art.
In addition to diagnostic methods, such as those described above, the present invention encompasses methods and compositions for typing and evaluating the prognosis of patients suffering from a disorder associated with aberrant activity or expression of GLUTX. The invention also encompasses methods and compositions for selecting an appropriate an treatment for disorders associated with inappropriate expression of GLUTX or inappropriate activity of GLUTX. The invention also includes compositions and methods for assessing the effectiveness of such treatments. For example, the nucleic acid molecules of the invention can be used as probes to classify cells in terms of their level of GLUTX expression and as primers or diagnostic PCR analysis which can be used to detect mutations, allelic variations, and regulator, defects in the GLUTX gene. Similarly, hose of ordinary skill in the art can use routine techniques to identify inappropriate activity of GLUTX, which can be observed in a variety of forms. Diagnostic kits for the practice of such methods are also provided.
The invention further encompasses transgenic animals that express GLUTX and recombinant xe2x80x9cknockoutxe2x80x9d animals that fail to express GLUTX. These animals can serve as new and useful models of disorders in which GLUTX is misexpressed.
The invention also features antagonists and agonists of GLUTX that can inhibit or enhance, respectively, one or more of the biological activities of GLUTX, e.g., the ability to act as a transporter for certain sugars. Suitable antagonists can include small molecules (i.e., molecules with a molecular weight below about 500), large molecules (i.e., molecules with a molecular weight above about 500), antibodies that specifically bind and xe2x80x9cneutralizexe2x80x9d GLUTX (as described below), and nucleic acid molecules that interfere with transcription or translation of GLUTX (e.g., antisense nucleic acid molecules and ribozymes). Agonists of GTUTX also include small and large molecules, and antibodies other than neutralizing antibodies.
The invention features methods and compositions useful for identifying antagonists and agonists of a GLUTX biological activity. These methods entail measuring the activity of GLUTX in the presence and absence of a test compound.
The invention also features molecules that can increase or decrease the expression of GLUTX (e.g., by altering transcription or translation). Small molecules (as defined above), large molecules (as defined above), and nucleic acid molecules (e.g., antisense and ribozyme molecules) can be used to inhibit the expression of GLUTX. Other types of nucleic acid molecules (e.g., molecules that bind to GLUTX negative transcriptional regulatory sequences) can be used to Increase the expression of GLUTX.
Compounds that modulate the expression of GLUTX in a cell can be identified by comparing the level of expression of GLUTX in the presence of a selected compound with the level of expression of GLUTX in the absence of that compound. A difference in the level of GLUTX expression indicating that he selected compound modulates the expression of GLUTX in the cell. A comparable test for compounds that modulate the activity of GLUTX can be carried out by comparing the level of GLUTX activity in the presence and absence of the compound. Thus, the in
The invention features methods and compositions useful for identifying compounds which modulate GLUTX expression. These methods entail measuring the expression of GLUTX (at the transcriptional or translational level) in the presence and absence of a test compound.
Patients who have a disorder mediated by abnormal GLUTX activity can be treated by administration of a compound that alters the expression of GLUTX or the activity of GLUTX. When the objective is to decrease expression or activity, the compound administered can be a GLUTX antisense oligonucleotide or an antibody, such as a neutralizing antibody, that specifically binds GLUTX, respectively.
The preferred methods and materials are described below in examples which are meant to illustrate, not limit, the invention. Skilled artisans will recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by, one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.